CN106083829B - A kind of hepatitis C virus inhibitors, pharmaceutical composition and its application - Google Patents
A kind of hepatitis C virus inhibitors, pharmaceutical composition and its application Download PDFInfo
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Abstract
The present invention provides a kind of hepatitis C virus inhibitors, pharmaceutical composition and its application, the hepatitis C virus inhibitors for example formula (I) compound represented or its crystal form, pharmaceutically acceptable salt, hydrate or solvated compounds.The compound of the present invention has better hepatitis C virus albumen NS5A inhibitory activity, with more preferable pharmacodynamics/pharmacokinetics performance, the applicability of compound is good, highly-safe, can be used for preparing the drug for the treatment of infection with hepatitis C virus, has the good prospect of marketing.
Description
Technical field
The invention belongs to pharmaceutical technology field more particularly to a kind of hepatitis C virus inhibitors, pharmaceutical composition and its
Using.
Background technique
HCV (Hepatitis C Virus, Hepatitis C Virus) is a kind of RNA virus, belongs to flaviviridae
Hepacivirus (Hepacivirus genus) in (Flaviviridae family).Wrap up HCV virus particle packet
The group of rna gene containing underlying stock encodes the special protein of virus-known to whole in single continual opening code-reading frame.
Opening code-reading frame includes about 9500 nucleotide and the huge polyprotein for encoding single about 3000 amino acid.Polyprotein packet
Include core albumen, wrap up albumen E1 and E2, embrane-associated protein P7 and non-structural protein NS2, NS3, NS4A, NS4B, NS5A and
NS5B。
Suo Feibuwei is first good medicine that can thoroughly cure hepatitis in a short time in the world at present.It is orally
Through lesion, the simple side effect very little of method are deep to be pursued by patient.Suo Feibuwei is produced by Ji Leadd B.V of the U.S., and 2013
Year listed in the U.S., through clinical test confirm can effective therapeutic gene 1,2,3,4 type hepatitis, including to liver transfer operation, liver cancer and
The clinical test of HCV/HIV-1 concurrent infection.This breakthrough is that global hepatitis patient brings Gospel.
HCV infection is related with progressive hepatopathy shape (including cirrhosis and hepatocellular carcinoma).(Achillion is public by Odalasvir
Hepatitis drug is taken charge of, be otherwise known as ACH-3102) it is a kind of for the new of potential treatment hepatitis C of Achillion company exploitation
Medicine is played a role by inhibiting hepatitis C virus protein NS5A, the state before being currently in registration.Its hepatitis cocktail
Therapy combination Suo Feibuwei is managed in the test for the treatment of I type genotype hepatitis patient when Ribavirin is not used
The second stage of clinical data thought can kill the virus of discovery in six weeks, this can reach most when being drug combination there are two types of institutes
Short treatment time and highest response rate.
The authorization of hepatitis new drug Suo Feibuwei is extended to Egypt by U.S.'s Ji Leadd B.V last year, and Egypt is the whole world third
The highest country of liver disease incidence.Then agree to that India produces Suo Feibuwei imitation medicine.So far, it is public that 8 India's pharmacy are shared
Department has taken the authorization of lucky moral, and Suo Feibuwei can be sold to global 91 developing countries (China is left out).Rope
Fei Buwei is in blank in China at present, is a large amount of demand on one side, is the listing of no medicine on one side, in face of such awkward awkward situation,
Some large hospitals are try to solve this problem by the overseas medical treatment of Video Remote.Some patients that can't wait, it has to examine
Worry, which is gone abroad, sees a doctor.Therefore, there is still a need for exploitations to have inhibitory activity or more preferable pharmacodynamics to hepatitis C virus protein NS5A for this field
The compound of performance.
Summary of the invention
Against the above technical problems, it the invention discloses a kind of hepatitis C virus inhibitors, pharmaceutical composition and its answers
With with better hepatitis C virus albumen NS5A inhibitory activity and/or with more preferable pharmacodynamics/pharmacokinetics performance.
In this regard, the technical solution adopted by the present invention are as follows:
A kind of hepatitis C virus inhibitors, the diaminopyrimidine compounds as shown in formula (I) or its crystal form, pharmaceutically
Acceptable salt, hydrate or solvated compounds,
Wherein, R1、R2、R3、R4、R5、R6、R7、R8、R9、R10、R11、R12、R13、R14、R15、R16、R17、R18、R19、R20、R21、
R22、R23、R24、R25、R26、R27、R28、R29、R30、R31、R32、R33、R33、R34、R35、R36、R37、R38、R39、R40、R41、R42、R43、
R44、R45、R46、R47、R48、R49、R50、R51、R52、R53、R54、R55、R56、R57、R58、R59、R60、R61、R62、R63、R64、R65、R66、
R67、R68It is each independently hydrogen, deuterium, halogen or trifluoromethyl；
Additional conditions are R1、R2、R3、R4、R5、R6、R7、R8、R9、R10、R11、R12、R13、R14、R15、R16、R17、R18、R19、
R20、R21、R22、R23、R24、R25、R26、R27、R28、R29、R30、R31、R32、R33、R33、R34、R35、R36、R37、R38、R39、R40、R41、
R42、R43、R44、R45、R46、R47、R48、R49、R50、R51、R52、R53、R54、R55、R56、R57、R58、R59、R60、R61、R62、R63、R64、
R65、R66、R67And R68In at least one be deuterated or deuterium.
It adopts this technical solution, shape and volume of the deuterium in drug molecule are substantially the same with hydrogen, if drug molecule
Middle hydrogen is optionally replaced with deuterium, and deuterated drug generally can also retain original bioactivity and selectivity.Inventor's warp simultaneously
It crosses it is experimentally confirmed that the combination of carbon deuterium key is more more stable than the combination of C-H bond, the absorption, distribution, generation of some drugs can be directly affected
The attributes such as thank and drain, to improve the curative effect of drug, safety and tolerance.
Preferably, deuterium isotopic content of the deuterium in deuterated position is at least greater than natural deuterium isotopic content (0.015%),
It is preferably greater than 30%, even more preferably greater than 50%, even more preferably greater than 75%, even more preferably greater than 95%, even more preferably greater than 99%.
Specifically, R in the present invention1、R2、R3、R4、R5、R6、R7、R8、R9、R10、R11、R12、R13、R14、R15、R16、
R17、R18、R19、R20、R21、R22、R23、R24、R25、R26、R27、R28、R29、R30、R31、R32、R33、R33、R34、R35、R36、R37、R38、
R39、R40、R41、R42、R43、R44、R45、R46、R47、R48、R49、R50、R51、R52、R53、R54、R55、R56、R57、R58、R59、R60、R61、
R62、R63、R64、R65、R66、R67And R68Deuterium isotopic content is at least 5% in each deuterated position, is preferably greater than 10%, more preferably
Ground is greater than 15%, even more preferably greater than 20%, even more preferably greater than 25%, even more preferably greater than 30%, even more preferably greater than 35%, more preferably
Greater than 40%, even more preferably greater than 45%, even more preferably greater than 50%, even more preferably greater than 55%, even more preferably greater than 60%, more preferably greatly
In 65%, even more preferably greater than 70%, even more preferably greater than 75%, even more preferably greater than 80%, even more preferably greater than 85%, even more preferably greater than
90%, even more preferably greater than 95%, even more preferably greater than 99%.
Preferably, compound at least contains a D-atom in formula (I), and the number containing D-atom can be in 1 to 68
Any one.
As a further improvement of the present invention, R1、R2、R3、R4、R5And R6It is each independently deuterium or hydrogen.
In another preferred example, R1、R2、R3、R4、R5、R6It is deuterium.
As a further improvement of the present invention, R7、R8、R9、R10、R11、R12、R13、R14、R15、R16、R17、R18、R19、R20、
R21And R22It is each independently deuterium or hydrogen.
In another preferred example, R7、R8、R9、R10、R11、R12、R13、R14、R15、R16、R17、R18、R19、R20、R21、R22It is
Deuterium.
As a further improvement of the present invention, R7、R15It is deuterium.
As a further improvement of the present invention, R8、R9、R10、R11、R12、R13、R16、R17、R18、R19、R20、R21It is deuterium.
As a further improvement of the present invention, R14、R22It is deuterium.
As a further improvement of the present invention, R23、R24、R25、R26、R27、R28、R29、R30、R31、R32、R33、R33、R34、
R35、R36、R37、R38、R39、R40、R41、R42、R43、R44、R45、R46、R47And R48It is each independently deuterium or hydrogen.
In another preferred example, R23、R24、R25、R26、R27、R28、R29、R30、R31、R32、R33、R33、R34、R35、R36、R37、
R38、R39、R40、R41、R42、R43、R44、R45、R46、R47、R48It is deuterium.
As a further improvement of the present invention, R23、R24、R25、R26、R27、R28、R29、R30、R31、R32、R36、R37、R38、
R39、R40、R41、R42、R43、R44、R45It is deuterium.
As a further improvement of the present invention, R33、R34、R35、R46、R47、R48It is deuterium.
As a further improvement of the present invention, R49、R50、R51、R52、R53And R54It is each independently deuterium or hydrogen.
In another preferred example, R49、R50、R51、R52、R53、R54It is deuterium.
As a further improvement of the present invention, R55、R56、R57、R58、R59And R60It is each independently deuterium or hydrogen.
In another preferred example, R55、R56、R57、R58、R59、R60It is deuterium.
As a further improvement of the present invention, R61、R62、R63、R64、R65、R66、R67And R68It is each independently deuterium or hydrogen.
In another preferred example, R61、R62、R63、R64、R65、R66、R67、R68It is deuterium.
As a further improvement of the present invention, the compound is selected from following compounds or its pharmaceutically acceptable salt:
In another preferred example, the compound does not include non-deuterated compound.
The invention also discloses a kind of pharmaceutical compositions, containing pharmaceutically acceptable carrier and described as described above
Hepatitis C virus inhibitors or its crystal form, pharmaceutically acceptable salt, hydrate or solvate, stereoisomer, preceding
The pharmaceutical composition of medicine or isotopic variations.
As a further improvement of the present invention, the pharmaceutically acceptable carrier includes glidant, sweetener, dilution
Agent, preservative, dyestuff/colorant, flavoring reinforcing agent, surfactant, wetting agent, dispersing agent, disintegrating agent, suspending agent, stabilization
At least one of agent, isotonic agent, solvent or emulsifier.
As a further improvement of the present invention, described pharmaceutical composition be tablet, pill, capsule, pulvis, granule,
Paste, emulsion, suspending agent, solution, suppository, injection, inhalant, gelling agent, microballoon or aerosol.
The classical pathway for giving pharmaceutical composition of the present invention includes but is not limited to oral, rectum, saturating mucous membrane, through enteral administration,
Or part, percutaneous, sucking, parenteral, sublingual, intravaginal, it is intranasal, intraocularly, peritonaeum is interior, intramuscular, subcutaneous, intravenous administration.
It is preferred that oral administration or drug administration by injection.
Pharmaceutical composition of the invention can be manufactured using method well known in the art, such as conventional mixing method, dissolution method,
Granulation, dragee method processed, levigate method, emulsion process, freeze-drying etc..
It as a further improvement of the present invention, also include other reactive compounds, the reactive compound is immune adjusts
Save agent or antiviral agent compounds.
As a further improvement of the present invention, the immunomodulator is interferons medical compounds.
As a further improvement of the present invention, the antiviral agent compounds are Ribavirin, amantadine, NS5A
Other inhibitor, the unwindase in HCV life cycle, protease, polymerase, metalloproteinases or internal ribosome entry site
The inhibitor of target, wherein other inhibitor of the NS5A are that he is at least one of big for Lei Dipawei or Dacca.
The present invention also provides a kind of methods for preparing pharmaceutical composition, comprising steps of by pharmaceutically acceptable carrier
It is carried out with hepatitis C virus inhibitors as described above or its crystal form, pharmaceutically acceptable salt, hydrate or solvate
Mixing forms pharmaceutical composition.
The invention also discloses a kind of purposes of hepatitis C virus inhibitors as described above, it is characterised in that: is used for
Purposes in the drug of preparation treatment infection with hepatitis C virus.
The HCV includes its Multi-genotype and several genes hypotype, for example, 1a, 1b, 2a, 2b, 3a, 3b, 4a,
5a、6a。
It should be understood that above-mentioned each technical characteristic of the invention and having in below (eg embodiment) within the scope of the present invention
It can be combined with each other between each technical characteristic of body description, to form a new or preferred technical solution.As space is limited, exist
This no longer tires out one by one states.
Herein, unless otherwise instructed, " halogen " refers to F, Cl, Br and I.More preferably, halogen atom is selected from F, Cl and Br.
Herein, unless otherwise instructed, " deuterated " refers to one or more hydrogen in compound or group replaced deuterium；Deuterium
In generation, can be a substitution, two replace, polysubstituted or full substitution.Term " one or more deuterated " and " one or many deuterated "
It is used interchangeably.
Herein, unless otherwise instructed, " non-deuterated compound " refers to ratio containing D-atom not higher than the same position of natural deuterium
The compound of cellulose content (0.015%).
Pharmaceutically acceptable salt includes inorganic salts and organic salt.A kind of preferred salt is that the compounds of this invention and acid are formed
Salt.The acid for suitably forming salt includes but is not limited to: the inorganic acids such as hydrochloric acid, hydrobromic acid, hydrofluoric acid, sulfuric acid, nitric acid, phosphoric acid；
Formic acid, acetic acid, trifluoroacetic acid, propionic acid, oxalic acid, malonic acid, succinic acid, fumaric acid, maleic acid, lactic acid, malic acid, tartaric acid,
The organic acids such as citric acid, picric acid, benzoic acid, methanesulfonic acid, ethanesulfonic acid, p-methyl benzenesulfonic acid, benzene sulfonic acid, naphthalene sulfonic acids；And dried meat ammonia
The amino acid such as acid, phenylalanine, aspartic acid, glutamic acid.Another kind of preferred salt is the salt that the compounds of this invention and alkali are formed,
Such as alkali metal salt (such as sodium salt or sylvite), alkali salt (such as magnesium salts or calcium salt), ammonium salt (such as rudimentary alkanol ammonium salt
And other pharmaceutically acceptable amine salt), such as methylamine salt, ethylamine salt, propylamine salt, dimethyl amine salt, trismethylamine salt, two
Ethyl amine salt, triethyl amine salt, tert-butylamine salt, ethylenediamine salt, oxyethylamine salt, dihydroxy ethylamine salt, three oxyethylamine salt, Yi Jifen
The amine salt not formed by morpholine, piperazine, lysine.
Term " solvate " refers to that the compounds of this invention and solvent molecule are coordinated the complex to form special ratios." hydration
Object " refers to that the compounds of this invention and water carry out the complex of coordination formation.
Compared with prior art, the invention has the benefit that
First, the compound of the present invention has excellent inhibition to hepatitis C virus protein NS5A.
Second, metabolism of the compound in organism is changed by this technology of deuterate, makes compound that there is better medicine
For kinetic parameter characteristic.In such a case, it is possible to change dosage and form durative action preparation, improve applicability.
Third improves compound in animal body due to its deuterium isotope effect with the hydrogen atom in deuterium substituted compound
Drug concentration, improve curative effect of medication.
4th, with the hydrogen atom in deuterium substituted compound, certain metabolites can be inhibited, improve the safety of compound
Property.
Specific embodiment
The preparation method of formula (I) structural compounds of the present invention is described more particularly below, but these specific methods are not to this
Invention constitutes any restrictions.The compounds of this invention can also optionally will be describing or known in the art various in the present specification
Synthetic method combines and is easily made, such combination can by those skilled in the art in the invention easily into
Row.
In general, each reaction is usually in atent solvent, in room temperature to reflux temperature (such as 0 DEG C~100 in preparation flow
DEG C, preferably 0 DEG C~80 DEG C) under carry out.Reaction time is usually -60 hours 0.1 hour, preferably 0.5-24 hours.
Embodiment 1
A kind of hepatitis C virus inhibitors, the entitled two-d3- methyl of chemistry ((2S, 2 ' S)-((2S, 2 ' S, 3aS, 3a ' S,
7aS, 7a ' S)-2,2 '-(5,5 '-(16-4,6,10,12,13,15- hexene-5,11- diyls of tricyclic [8.8.2.24,7]) two
(1 hydrogen-benzimidazole -5,2- diyl)) two (- 1 hydrogen of octahydro-indoles -2,1- diyl)) two (3- methyl-1s-oxo-butanes -2,1-
Diyl) diurethane, i.e. compound T-1, molecular formula be as follows:
It is synthesized using following route:
The following steps are included:
1, -1 hydrogen of (2R, 3aS, 7aS) -1- (tertbutyloxycarbonyl) octahydro-indole-2-carboxylic acid (compound 1) synthesis；
L- octahydro indole-2-carboxylic acid (1.69g, 10mmol) is added in reaction flask, 30mL THF and 30mL distillation is added
The 7mL aqueous solution of NaOH (0.68g, 17mmol) is added dropwise under ice bath, finishes, is stirred to react 10min for water stirring and dissolving.It is added dropwise
(Boc)2O (2.84g, 13mmol) is warmed to room temperature reaction 12h.TLC is detected after completion of the reaction, water dilution is added, with methyl- tert fourth
Base ether abstraction impurity removal, water phase citric acid tune PH to acidity, ethyl acetate extract three times, merge organic phase, saturated common salt washing
It washs, Na2SO4It dries, filters and is concentrated to get 3.21g compound 1, yield 100%.LC-MS (APCI): m/z=268.3 (M-1)-。
2, (2R, 3aS, 7aS)-tert-butyl -2- ((2- amino -4- bromophenyl) carbamyl) -1 hydrogen of octahydro-indoles -1- carboxylic acid
The synthesis of ester (compound 2)；
Compound 1 (2.69g, 10mmol) and 4- bromine o-phenylenediamine (2.244g, 12mmol) are added in reaction flask, is added
The dissolution of 60mL acetonitrile, addition condensing agent EDCI (1- ethyl-(3- dimethylaminopropyl) carbodiimide hydrochloride) (2.3g,
12mmol), n,N-diisopropylethylamine (1.68g, 13mmol) is added dropwise at 0 DEG C, finishes, is warmed to room temperature and is stirred to react 1 hour, point
Plate detects after completion of the reaction, and concentration removes half acetonitrile, and 120mL water is added, is stirred overnight at room temperature.Filtering, filter cake are washed with water
It washs, is dried in vacuo to obtain product 3.6g, yield 82.2%.LC-MS (APCI): m/z=439.1 (M+1)+。
3, -1 carboxylate of (2R, 3aS, 7aS)-tert-butyl -2- (the bromo- 1 hydrogen-benzimidazolyl-2 radicals-yl of 6-) -1 hydrogen of octahydro-indoles
The synthesis of (compound 3)；
Compound 2 (2.19g, 5mmol) is used into 10mL acetic acid, 65 DEG C is heated to and is stirred to react 3 hours, TLC detection
It being concentrated after completion of the reaction and removes solvent, ethyl acetate dilution is added, be added with stirring 2mL ammonium hydroxide, tune PH to alkalescent is washed,
Water phase is extracted with ethyl acetate, and merges organic phase, and saturated common salt water washing is concentrated, and column chromatographic purifying obtains 2.18g compound 3,
Yield 100%.LC-MS (APCI): m/z=421.1 (M+1)+。
4, (2R, 3aS, 7aS)-tert-butyl -2- (6- (4,4,5,5- tetramethyl -1,3,2- dioxy borine -2- base) -1 hydrogen -
Benzimidazolyl-2 radicals-yl) -1 hydrogen of octahydro-indoles -1 carboxylate (compound 4) synthesis；
In dry reaction flask be added compound 3 (2.1g, 5mmol), connection boric acid pinacol ester (1.9g, 7.5mmol),
Pd(dppf)Cl2The anhydrous dioxy of 21mL is added under nitrogen protection for (0.366g, 1.5mmol) and potassium acetate (1.47g, 15mmol)
Six rings are heated to 85 DEG C and are stirred to react 4 hours, and ethyl acetate dilution is added in TLC detection after completion of the reaction, and active carbon decoloring is added
1 hour, filtering was concentrated to give oily liquids, column chromatographic purifying obtains 3.1g compound 4, yield 100%.LC-MS(APCI):m/z
=468.7 (M+1)+。
5, the synthesis of 4,16- dibromo [2.2] polyxylene (compound 5)；
Bromine (0.863g, 20.3mmol) and iron powder (48mg, 1.86mmol) are added in reaction flask, 16ml dichloro is added
Methane is stirred to react 1 hour at room temperature.The 3mL dichloromethane solution of raw material dimeric p-xylene (2.08g, 10mmol) is added,
It is heated to flowing back, the 5ml dichloromethane solution of 2.4g bromine is slowly added dropwise, finishes, back flow reaction 3 hours, be down to room temperature reaction
Overnight.Methylene chloride dilution is added, successively with 5% sodium thiosulfate, water, saturated common salt water washing, anhydrous sodium sulfate is dry,
Filtering and concentrating, re crystallization from toluene obtain 1.56g compound 5, yield 42.6%.1H NMR(300MHz,CDCl3):δ(ppm):
2.79-3.00 (m, 4H), 3.10-3.21 (m, 2H), 3.44-3.54 (m, 2H), 6.44 (d, J=8.0Hz, 2H), 6.51 (d, J
=2.0Hz, 2H), 7.14 (dd, J=8.0Hz, 2.0Hz, 2H).
6, (2S, 2 ' S, 3aS, 3a ' S, 7aS, 7a ' S)-di-t-butyl -2,2 '-(5,5 '-(tricyclic [8.8.2.24,7] ten
Six -4,6,10,12,13,15- hexene -5,11- diyls) two (1 hydrogen-benzimidazole -5,2- diyl) two (- 1 hydrogen of octahydro-indoles -
1- carboxylate) (compound 6) synthesis；
Compound 4 (2.3g, 4.92mmol), compound 5 (0.72g, 1.97mmol), cesium carbonate are added in reaction flask
(1.61g, 4.92mmol) and Pd (PPh3)4(1.115g, 0.1mmol) 18mL DMF and 1ml water is added under nitrogen protection, is added
It is reacted 3 hours to 130 DEG C, white solid, mistake is precipitated in Filtration of catalyst, addition distilled water after completion of the reaction for contact plate detection
It is filtered dry dry crude product, obtains 460mg compound 6, yield 26.3% after column chromatographic purifying.LC-MS (APCI): m/z=888.7 (M
+1)+。
7, (2S, 2 ' S, 3aS, 3a ' S, 7aS, 7a ' S) -2,2 '-(5,5 '-(tricyclic [8.8.2.24,7] 16-4,6,10,
12,13,15- hexene -5,11- diyl) two (1 hydrogen-benzimidazole -5,2- diyl) two (- 1 hydrogen of octahydro-indoles) (compounds 7)
Synthesis；
Compound 6 (460mg, 0.518mmol) is dissolved in the mixed solvent of 4mL methylene chloride and 1mL methanol, is added at 0 DEG C
Enter 2.33ml hydrogen chloride dioxane, be warmed to room temperature reaction 1 hour, after TLC is detected, concentration removes solvent, obtains compound 7
Crude product is not required to purifying and is directly thrown into next step.
8, the synthesis of chloro-carbonic acid-d3- methyl esters (compound 8)；
Triphosgene (2.67g, 9mmol) is dissolved in 20ml toluene, at 0 DEG C be added dropwise deuterated methanol (1.08g, 30mmol) and
The 20ml toluene solution of triethylamine (3.34g, 33mmol), finishes, and is warmed to room temperature reaction 1 hour.Reaction solution is washed with ice water
Three times, anhydrous sodium sulfate dry, filter compound 8 toluene solution, be directly thrown into next step react.
9, d3- methoxycarbonyl group-Valine (compound 9) synthesis；
Valine (2.81g, 24mmol) is dissolved in 20mL dioxane, addition 1N sodium hydroxide (2.9g,
72mmol) aqueous solution, the toluene solution of step gained compound 8 in dropwise addition, reaction is overnight at room temperature.TLC is detected after completion of the reaction
Dilute hydrochloric acid tune PH is added to acidity, is extracted with ethyl acetate three times, merges organic phase, saturated common salt water washing, anhydrous sodium sulfate
It dries, filters and is concentrated to get 3.56g compound 9, yield 83.3%.LC-MS (APCI): m/z=179.1 (M+1)+。
10, two-d3- methyl ((2S, 2 ' S)-((2S, 2 ' S, 3aS, 3a ' S, 7aS, 7a ' S) -2,2 '-(5,5 '-(tricyclics
[8.8.2.24,7] 16-4,6,10,12,13,15- hexene-5,11- diyls) two (1 hydrogen-benzimidazole-5,2- diyls)) two
(- 1 hydrogen of octahydro-indoles-2,1- diyl)) two (3- methyl-1-oxo-butanes-2,1- diyl) diurethanes, i.e. compound
The synthesis of T-1；
Compound 9 (98.53mg, 0.553mmol), HOBT (74.7mg, 0.553mmol) and EDCI are added in reaction flask
(106.1mg, 0.553mmol) is dissolved with 3mL acetonitrile, reacts 5 minutes the solution 1 after being activated at room temperature.By compound 7
(200mg, 0.24mmol) is dissolved in 5mL DMF, and DIPEA (223.3mg, 1.73mmol) is added and obtains solution 2.Solution 1 is added
Enter into solution 2, be stirred to react at room temperature 3 hours, distilled water is added in TLC detection after completion of the reaction, and white solid is precipitated, and takes out
Filter, filtration cakes torrefaction rear pillar chromatographic purifying obtain 120mg compound T-1, yield 49.8%.
1H NMR(300MHz,CDCl3) δ 10.87 (s, 2H), 7.94 (dd, J=31.7,9.3Hz, 2H), 7.61-7.30
(m, 4H), 6.82-6.54 (m, 6H), 5.54-5.37 (m, 4H), 4.27 (dd, J=20.0,10.6Hz, 4H), 3.60-3.23
(m, 4H), 3.01 (d, J=5.7Hz, 2H), 2.95 (s, 2H), 2.88 (s, 2H), 2.77 (s, 4H), 2.51 (s, 2H), 2.41-
2.28 (m, 2H), 2.02 (ddd, J=35.3,20.2,9.3Hz, 4H), 1.76 (s, 10H), 0.97 (dt, J=11.6,5.7Hz,
6H), 0.85 (dd, J=6.9,3.6Hz, 6H).LC-MS (APCI): m/z=1008.3 (M+1)+。
Embodiment 2
Dimethyl ((2S, 2 ' S)-((2S, 2 ' S, 3aS, 3a ' S, 7aS, 7a ' S) -2,2 '-(5,5 '-(tricyclic [8.8.2.24 ,7] 16-4,6,10,12,13,15- hexene-5,11- diyls) two (1 hydrogen-benzimidazole-5,2- diyls)) two (- 1 hydrogen of octahydro-
Indoles-2,1- diyl)) two (3-d3- methyl-1-oxo-d5- butane-2,1- diyl) diurethanes, i.e. compound T-2,
Its molecular formula is
It is synthesized using following route:
On the basis of embodiment 1, specifically includes the following steps:
1, the synthesis of methylchloroformate (compound 10)；
Triphosgene (0.3g, 1mmol) is dissolved in 2mL toluene, methanol (96.12mg, 3mmol) and three second are added dropwise at 0 DEG C
The 2mL toluene solution of amine (334mg, 3.3mmol), finishes, and is warmed to room temperature reaction 1 hour.Reaction solution is washed three times with ice water,
Anhydrous sodium sulfate dry, filter compound 10 toluene solution, be directly thrown into next step react.
2, the synthesis of methoxycarbonyl group-d8-L- valine (compound 11)；
Deuterated Valine (300mg, 2.4mmol) is dissolved in 3mL dioxane, addition 1N sodium hydroxide (288mg,
7.2mmol) aqueous solution, the toluene solution of step gained compound 8 in dropwise addition, reaction is overnight at room temperature.TLC is detected after completion of the reaction
Dilute hydrochloric acid tune pH is added to acidity, is extracted with ethyl acetate three times, merges organic phase, saturated common salt water washing, anhydrous sodium sulfate
It dries, filters and is concentrated to get 280mg compound 11, yield 63.8%.LC-MS (APCI): m/z=184.1 (M+1)+。
3, dimethyl ((2S, 2 ' S)-((2S, 2 ' S, 3aS, 3a ' S, 7aS, 7a ' S) -2,2 '-(5,5 '-(tricyclics
[8.8.2.24,7] 16-4,6,10,12,13,15- hexene-5,11- diyls) two (1 hydrogen-benzimidazole-5,2- diyls)) two
(- 1 hydrogen of octahydro-indoles-2,1- diyl)) (change of two (3-d3- methyl-1-oxo-d5- butane-2,1- diyl) diurethanes
Close object T-2) synthesis；
Compound 11 (140mg, 0.764mmol), HOBT (103.2mg, 0.764mmol) and EDCI are added in reaction flask
(146.1mg, 0.764mmol) is dissolved with 2mL acetonitrile, reacts 5 minutes the solution 1 after being activated at room temperature.By compound 7
(276.53mg, 0.332mmol) is dissolved in 5mL DMF, and DIPEA (309mg, 2.39mmol) is added and obtains solution 2.By solution 1
It is added in solution 2, is stirred to react at room temperature 3 hours, distilled water is added in TLC detection after completion of the reaction, and white solid is precipitated, and takes out
Filter, filtration cakes torrefaction rear pillar chromatographic purifying obtain 90mg compound T-2, yield 26.9%.1H NMR(300MHz,CDCl3)δ10.86
(d, J=13.5Hz, 2H), 8.02-7.83 (m, 2H), 7.42 (ddd, J=31.6,25.6,9.6Hz, 4H), 6.68 (t, J=
18.7Hz, 6H), 5.52 (d, J=11.7Hz, 2H), 5.46-5.31 (m, 2H), 4.31 (d, J=5.9Hz, 2H), 3.74 (d, J
=15.1Hz, 6H), 3.59-3.25 (m, 4H), 3.02 (s, 2H), 2.95 (s, 2H), 2.88 (s, 2H), 2.77 (s, 4H), 2.51
(s, 2H), 2.36 (s, 2H), 1.93 (d, J=12.7Hz, 2H), 1.85-1.69 (m, 10H).LC-MS (APCI): m/z=
1018.4(M+1)+。
Embodiment 3
Dimethyl ((2S, 2 ' S)-((2S, 2 ' S, 3aS, 3a ' S, 7aS, 7a ' S) -2,2 '-(5,5 '-(tricyclic [8.8.2.24 ,7] 16-4,6,10,12,13,15- hexene-5,11- diyls) two (1 hydrogen-d3- benzimidazole-5,2- diyls)) two (octahydros-1
Hydrogen-indoles-2,1- diyl)) two (3- methyl-1-oxo-butane-2,1- diyl) diurethanes, i.e. compound T-3, point
Minor is as follows:
It synthesizes to obtain using following route:
On the basis of embodiment 1, comprising the following steps:
1, the synthesis of the bromo- tri- deuterated o-phenylenediamine (compound 12) of 3,5,6- of 4-；
4- bromine o-phenylenediamine (374mg, 2mmol), concentrated hydrochloric acid (0.33mL, 4mmol) and 5mL weight are added in reaction flask
Water, stirring and dissolving are placed in microwave reactor, and 140 DEG C are reacted 0.5 hour, are cooled to room temperature, are added 1N sodium hydroxide tune PH extremely
Alkalinity is extracted with ethyl acetate three times, merges organic phase, and saturated common salt water washing is concentrated to give crude product, and column chromatographic purifying obtains
365mg compound 12, yield 97.6%.LC-MS (APCI): m/z=191.4 (M+1)+。
2, (2R, 3aS, 7aS)-tert-butyl -2- ((the deuterated phenyl of the bromo- 3,5,6- tri- of 2- amino -4-) carbamyl) octahydro -1
The synthesis of hydrogen-indoles -1- carboxylate (compound 13)；
Compound 1 (1.62g, 6mmol) and compound 12 (1.37g, 7.2mmol) are added in reaction flask, 36mL is added
Acetonitrile dissolution, is added condensing agent EDCI (1.38g, 7.2mmol), and DIPEA (1.01g, 7.8mmol) is added dropwise at 0 DEG C, finishes, and rises
To reaction 1 hour is stirred at room temperature, TLC is detected after completion of the reaction, and concentration removes half acetonitrile, and 72mL water is added, stirs at room temperature
Overnight.Filtering, filter cake are washed with water, and are dried in vacuo to obtain product 2.19g, yield 81%.LC-MS (APCI): m/z=442.1 (M+
1)+。
3, (2R, 3aS, 7aS)-tert-butyl -2- (deuterated -1 hydrogen-benzimidazolyl-2 radicals-yl of the bromo- 5,7,8- tri- of 6-) octahydro -1
The synthesis of -1 carboxylate of hydrogen-indoles (compound 14)；
Compound 13 (2.19g, 5mmol) is used into 10mL acetic acid, 65 DEG C is heated to and is stirred to react 3 hours, TLC detection
It being concentrated after completion of the reaction and removes solvent, ethyl acetate dilution is added, be added with stirring 2mL ammonium hydroxide, tune PH to alkalescent is washed,
Water phase is extracted with ethyl acetate, and merges organic phase, and saturated common salt water washing is concentrated, and column chromatographic purifying obtains 2.18g compound
14, yield 100%.LC-MS (APCI): m/z=424.2 (M+1)+。
4, (2R, 3aS, 7aS)-tert-butyl -2- (6- (4,4,5,5- tetramethyl -1,3,2- dioxy borine -2- base) -5,7,
Deuterated -1 hydrogen-benzimidazolyl-2 radicals-yl of 8- tri-) -1 hydrogen of octahydro-indoles -1 carboxylate (compound 15) synthesis；
In dry reaction flask be added compound 14 (2.1g, 5mmol), connection boric acid pinacol ester (1.9g,
7.5mmol)、Pd(dppf)Cl221mL is added under nitrogen protection in (0.366g, 1.5mmol) and potassium acetate (1.47g, 15mmol)
Anhydrous dioxane is heated to 85 DEG C and is stirred to react 4 hours, and ethyl acetate dilution is added in TLC detection after completion of the reaction, is added and lives
Property carbon decoloring 1 hour, filtering, be concentrated to give oily liquids, column chromatographic purifying obtains 3.1g compound 15, yield 100%.LC-MS
(APCI): m/z=471.2 (M+1)+。
5, (2S, 2 ' S, 3aS, 3a ' S, 7aS, 7a ' S)-di-t-butyl -2,2 '-(5,5 '-(tricyclic [8.8.2.24,7] ten
Six -4,6,10,12,13,15- hexene -5,11- diyls) two (deuterated -1 hydrogen-benzimidazole -5,2- diyls of 5,7,8- tri-) two (eight
- 1 hydrogen of hydrogen-indoles -1- carboxylate) (compound 16) synthesis；
Compound 15 (1.85g, 3.93mmol), compound 5 (0.58g, 1.57mmol), cesium carbonate are added in reaction flask
(1.28g, 3.92mmol) and Pd (PPh3)4(90.7mg, 0.08mmol) 15mL DMF and 1mL water is added under nitrogen protection, adds
Enter to 130 DEG C and react 3 hours, white solid, mistake is precipitated in Filtration of catalyst, addition distilled water after completion of the reaction for TLC detection
It is filtered dry dry crude product, obtains 480mg compound 16, yield 34.3% after column chromatographic purifying.LC-MS (APCI): m/z=894.5
(M+1)+。
6, (2S, 2 ' S, 3aS, 3a ' S, 7aS, 7a ' S) -2,2 '-(5,5 '-(tricyclic [8.8.2.24,7] 16-4,6,10,
12,13,15- hexene -5,11- diyl) two (deuterated -1 hydrogen-benzimidazole -5,2- diyl of 5,7,8- tri-) two (- 1 hydrogen-Yin of octahydro
Diindyl) (compound 17) synthesis；
Compound 16 (480mg, 0.537mmol) is dissolved in the mixed solvent of 4mL methylene chloride and 1mL methanol, is added at 0 DEG C
Enter 2.0mL hydrogen chloride dioxane, be warmed to room temperature reaction 1 hour, after TLC is detected, concentration removes solvent, obtains compound 17
Crude product is not required to purifying and is directly thrown into next step.
7, dimethyl ((2S, 2 ' S)-((2S, 2 ' S, 3aS, 3a ' S, 7aS, 7a ' S) -2,2 '-(5,5 '-(tricyclics
[8.8.2.24,7] 16-4,6,10,12,13,15- hexene-5,11- diyls) two (1 hydrogen-d3- benzimidazole-5,2- diyls))
Two (- 1 hydrogen of octahydro-indoles-2,1- diyl)) two (3- methyl-1-oxo-butane-2,1- diyl) diurethanes, i.e. chemical combination
The synthesis of object T-3；
In reaction flask be added Moc-L- valine (388.9mg, 2.22mmol), HOBT (300mg, 2.22mmol) and
EDCI (425.6mg, 2.22mmol) is dissolved with 5mL acetonitrile, reacts 5 minutes the solution 1 after being activated at room temperature.By chemical combination
Object 17 (802.2mg, 0.965mmol) is dissolved in 10mL DMF, and DIPEA (898.3mg, 6.95mmol) is added and obtains solution 2.It will
Solution 1 is added in solution 2, is stirred to react at room temperature 3 hours, and distilled water is added in TLC detection after completion of the reaction, and it is solid that white is precipitated
Body, filters, and filtration cakes torrefaction rear pillar chromatographic purifying obtains 400mg compound T-3, yield 41.2%.
1H NMR(300MHz,CDCl3) δ 10.85 (d, J=13.0Hz, 2H), 6.77-6.60 (m, 6H), 5.53-5.37
(m, 4H), 4.44-4.13 (m, 4H), 3.77 (d, J=21.4Hz, 7H), 3.45 (dd, J=21.0,8.0Hz, 4H), 3.03 (s,
2H), 2.96 (s, 2H), 2.86 (d, J=18.3Hz, 2H), 2.71 (d, J=42.0Hz, 4H), 2.52 (s, 2H), 2.37 (s,
2H), 2.08-1.90 (m, 4H), 1.72 (s, 12H), 0.98 (dt, J=10.6,5.5Hz, 6H), 0.89-0.83 (m, 6H).LC-
MS (APCI): m/z=1008.3 (M+1)+。
Embodiment 4
Dimethyl ((2S, 2 ' S)-((2S, 2 ' S, 3aS, 3a ' S, 7aS, 7a ' S) -2,2 '-(5,5 '-(tricyclic [8.8.2.24 ,7] 16-5,7,8,12,13,15-d6-4,6,10,12,13,15- hexene-5,11- diyls) two (1 benzimidazole-5 hydrogen-d3-,
2- diyl)) two (- 1 hydrogen of octahydro-indoles-2,1- diyl)) two (3- methyl-1-oxo-butane-2,1- diyl) diamino acids
Ester, i.e. compound T-4, molecular formula are as follows:
It synthesizes to obtain using following route:
The following steps are included:
1, the synthesis of bis- bromo- 5,7,8,12,13,15-d6 [2.2] polyxylene (compound 18) of 4,16-；
Raw material compound 5 (109.8mg, 0.3mmol), MoCl are added in reaction flask5(8.2mg, 0.03mmol) and 2mL
Deuterated benzene reacts 1.5 hours at 80 DEG C of microwave, is cooled to room temperature, Filtration of catalyst, is concentrated to give crude product, column chromatographic purifying
Obtain compound 100mg compound 18, yield: 91.7%.LC-MS (APCI): m/z=373.3 (M+1)+。
2, (2S, 2 ' S, 3aS, 3a ' S, 7aS, 7a ' S)-di-t-butyl -2,2 '-(5,5 '-(tricyclic [8.8.2.24,7] ten
Six -5,7,8,12,13,15-d6-4,6,10,12,13,15- hexene -5,11- diyls) two (1 hydrogen-benzimidazole -5,2- diyls)
The synthesis that two (- 1 hydrogen of octahydro-indoles -1- carboxylate) (compounds 19) use；
Compound 4 (1.85g, 3.93mmol), compound 18 (0.58g, 1.57mmol), cesium carbonate are added in reaction flask
15mL DMF and 1mL water is added under nitrogen protection, adds by (1.28g, 3.92mmol) and Pd (PPh3) 4 (90.7mg, 0.08mmol)
Enter to 130 DEG C and react 3 hours, white solid, mistake is precipitated in Filtration of catalyst, addition distilled water after completion of the reaction for TLC detection
It is filtered dry dry crude product, obtains 480mg compound 19, yield 34.1% after column chromatographic purifying.LC-MS (APCI): m/z=894.5
(M+1)+。
3, (2S, 2 ' S, 3aS, 3a ' S, 7aS, 7a ' S) -2,2 '-(5,5 '-(tricyclic [8.8.2.24,7] 16-5,7,8,
12,13,15-d6-4,6,10,12,13,15- hexene -5,11- diyl) two (1 hydrogen-benzimidazole -5,2- diyl) two (octahydros -1
Hydrogen-indoles) (compound 20) synthesis；
Compound 16 (480mg, 0.537mmol) is dissolved in the mixed solvent of 4mL methylene chloride and 1mL methanol, is added at 0 DEG C
Enter 2.0mL hydrogen chloride dioxane, be warmed to room temperature reaction 1 hour, after TLC is detected, concentration removes solvent, obtains compound 20
Crude product is not required to purifying and is directly thrown into next step.
4, dimethyl ((2S, 2 ' S)-((2S, 2 ' S, 3aS, 3a ' S, 7aS, 7a ' S) -2,2 '-(5,5 '-(tricyclics
[8.8.2.24,7] 16-5,7,8,12,13,15-d6-4,6,10,12,13,15- hexene-5,11- diyls) two (1 hydrogen-d3- benzene
And imidazoles-5,2- diyl)) two (- 1 hydrogen of octahydro-indoles-2,1- diyl)) two (3- methyl-1-oxo-butane-2,1- diyls) two
Carbamate, i.e. compound T-4) synthesis；
Moc-L- valine (98.53mg, 0.553mmol), HOBT (74.7mg, 0.553mmol) are added in reaction flask
It with EDCI (106.1mg, 0.553mmol), is dissolved with 3mL acetonitrile, reacts 5 minutes the solution 1 after being activated at room temperature.It will change
It closes object 20 (200mg, 0.24mmol) to be dissolved in 5mL DMF, DIPEA (223.3mg, 1.73mmol) is added and obtains solution 2.It will be molten
Liquid 1 is added in solution 2, is stirred to react at room temperature 3 hours, and distilled water is added in TLC detection after completion of the reaction, and it is solid that white is precipitated
Body, filters, and filtration cakes torrefaction rear pillar chromatographic purifying obtains 78mg compound T-4, yield 32.4%.
1H NMR(300MHz,CDCl3) δ 10.88 (s, 2H), 8.00 (d, J=9.8Hz, 1H), 7.88 (s, 1H), 7.62-
7.33 (m, 4H), 5.54-5.38 (m, 4H), 4.41-4.22 (m, 4H), 3.75 (d, J=16.2Hz, 6H), 3.44 (dd, J=
24.4,10.4Hz, 4H), 3.03 (s, 2H), 2.96 (s, 2H), 2.87 (d, J=9.3Hz, 2H), 2.79 (d, J=6.6Hz,
4H), 2.52 (s, 2H), 2.36 (d, J=11.9Hz, 2H), 1.94 (d, J=14.3Hz, 4H), 1.76 (d, J=10.7Hz,
11H),1.02–0.94(m,6H),0.89–0.81(m,6H).LC-MS (APCI): m/z=1008.7 (M+1)+。
Embodiment 5
Biological evaluation is carried out to the compound of above embodiments.
In order to verify effect of the compound as described herein to HCV, inventor uses HCV Replicate Sub-system (HCV
Replicon System) it is used as evaluation model.Since Science1999 is reported for the first time, HCV Replicate Sub-system at
For study HCV rna replicon, pathogenic and viral persistence one of the most important instruments, such as utilize replicon successfully
Ground demonstrates 5'-NCR Minimum Area necessary to HCV rna replicon, and HCV Replicate Sub-system has been successfully used as resisting
The evaluation model of virus drugs.The present inventor is according to Science, 1999,285 (5424), 110-3, and
J.Virol, 2003,77 (5), method described in 3007-19 are verified.
(1) detection compound HCV-Ab IgG 1a and 1b genocopy activity
Using HCV-1a and HCV-1b stable transfection replicon cell detection compound hepatitis c virus genotype 1a and
The inhibitory activity of 1b replicon.This experiment will be using NS5A inhibitor BMS-790052 as positive reference compound.
Step 1: 1:3 is carried out to compound and is serially diluted 8 concentration points, duplicate hole is added in 96 orifice plates.DMSO is set
For nothing plus compound control.DMSO ultimate density in cell culture fluid is 0.5%.
Step 2: HCV-1a and 1b cell is suspended in respectively in the culture solution containing 10%FBS, 8,000 thin with every hole
The density kind of born of the same parents is into containing compound 96 orifice plate.Cell is in 5%CO2, cultivate 3 days under the conditions of 37 DEG C.
Step 3: with CellTiter-Fluor (Promega) measurement compound to GT1b replicon cell toxicity.
Step 4: luciferase assay compound antihepatitis C virus activity is detected with Bright-Glo (Promega).
Step 5: using GraphPad Prism software analysis data, and matched curve simultaneously calculates EC50Value and CC50Value.
Analytical calculation EC50 value and CC50 value are carried out according to step as above to the compound of 1~embodiment of embodiment 4, as a result
As shown in table 1.
(2) measurement compound anti-infection property Hepatitis C virus (GT2a) activity
Document (A novel luciferase and GFP dual reporter has been reported in infection model reference
virus for rapid and convenient evaluation of HCV replication.Virus Res
2011.155:406).With HCVcc (MOI=0.2) infect Huh-7.5.1 cell after, compound handle 3 days (8 concentration, 3 times
Dilution, duplicate hole).Antiviral activity will be with fluorescein enzymatic assays.Compound is measured to Huh7.5.1 cell activity simultaneously.This
Experiment will be using NS5A inhibitor BMS-790052 as positive reference compound.EC is calculated using GraphPad software50。
Analytical calculation EC is carried out according to above-mentioned steps to the compound of 1~embodiment of embodiment 450, the results are shown in Table 1.
The biological evaluation analytical table of 1 1~embodiment of embodiment 4 of table
As shown in table 1, it was demonstrated that the compound of the present invention can inhibit multiple genotype of HCV, and by inhibiting HCV NS5A
The mechanism of albumen has played superior anti-hepatitis C virus effect.
(3) metabolic stability is evaluated
Microsomal assay: people's hepatomicrosome: 0.5mg/mL, Xenotech；Rat liver microsomes: 0.5mg/mL,
Xenotech；Coenzyme (NADPH/NADH): 1mM, Sigma Life Science；Magnesium chloride: 5mM, 100mM phosphate buffer
(pH 7.4).
The preparation of stock solution: precision weighs the powder of a certain amount of COMPOUNDS EXAMPLE 1-4, and is dissolved to respectively with DMSO
5mM。
The preparation of phosphate buffer (100mM, pH7.4): take the 0.5M potassium dihydrogen phosphate 150mL for preparing in advance and
The 0.5M dipotassium hydrogen phosphate solution of 700mL mixes, then adjusts mixed liquor pH value to 7.4 with 0.5M dipotassium hydrogen phosphate solution, uses
It is preceding to dilute 5 times with ultrapure water, magnesium chloride is added, phosphate buffer (100mM) is obtained, wherein potassium phosphate containing 100mM, 3.3mM
Magnesium chloride, pH 7.4.
It prepares NADPH regenerative system solution and (contains 6.5mM NADP, 16.5mM G-6-P, 3U/mL G-6-P D, 3.3mM
Magnesium chloride), using it is preposition in it is wet on ice.
Prepare terminate liquid: the acetonitrile containing 50ng/mL Propranolol Hydrochloride and 200ng/mL orinase (internal standard) is molten
Liquid.It takes 25057.5 μ L phosphate buffers (pH7.4) into 50mL centrifuge tube, is separately added into 812.5 μ L people's hepatomicrosomes, mix
It is even, obtain the hepatomicrosome dilution that protein concentration is 0.625mg/mL.Take 25057.5 μ L phosphate buffers (pH7.4) extremely
In 50mL centrifuge tube, 812.5 μ L SD rat liver microsomes are separately added into, are mixed, the liver that protein concentration is 0.625mg/mL is obtained
Microsome dilution.
The incubation of sample: being diluted to 0.25mM for the stock solution of respective compound with the aqueous solution containing 70% acetonitrile respectively,
It is spare as working solution.It takes people's hepatomicrosome of 398 μ L or rat liver microsomes dilution that 96 holes are added respectively to be incubated in plate
(N=2), it is separately added into the working solution of 2 μ L 0.25mM, mixes.
The measurement of metabolic stability: the terminate liquid of 300 μ L pre-cooling is added in every hole of 96 hole deep-well plates, is placed in ice
On, as termination plate.96 holes are incubated for plate and NADPH regenerative system is placed in 37 DEG C of water baths, 100 revs/min of concussions are incubated in advance
5min.80 μ L Incubating Solutions addition termination plate is taken out from the every hole of plate is incubated for, mixes, supplements 20 μ L NADPH regenerative system solution, make
For 0min sample.Again to the NADPH regenerative system solution for being incubated for 80 μ L of the every hole addition of plate, starting reaction starts timing.Correspondingization
The reaction density for closing object is 1 μM, protein concentration 0.5mg/mL.When reacting 10,30,90min, 100 μ L is respectively taken to react
Liquid is added in termination plate, and vortex 3min terminates reaction.Termination plate is centrifuged 10min under the conditions of 5000 × g, 4 DEG C.Take 100 μ L
Supernatant is mixed to being previously added in 96 orifice plates of 100 μ L distilled water, carries out sample analysis using LC-MS/MS.
Data analysis: by LC-MS/MS system detection respective compound and interior target peak area, calculate compound with it is interior
Mark peak area ratio.Slope is measured by the natural logrithm of the percentage of compound surplus and time mapping, and according to following
Formula calculates t1/2And CLint, wherein V/M is equal to 1/ protein concentration.
The compound of embodiment 1-4 is analyzed according to above-mentioned steps, the results are shown in Table 2.
The measurement result table of the compound metabolism stability of 2 embodiment 1-4 of table
As shown in table 2, the compounds of this invention is all shown excellent in people's hepatomicrosome and rat liver microsomes experiment
Metabolic stability.
(4) pharmacokinetics in rats is tested
Experiment purpose: after research rat gives Odalasvir, compound T-1, the medicine of the compounds of this invention is investigated for power
Scholarship and moral conduct is.
Experimental animal:
Type and strain: SD rat grade: SPF grades
Gender and quantity: male, 6
Weight range: 180~220g (actual weight range is 187~197g)
Source: the western Poole Bi Kai experimental animal Co., Ltd in Shanghai
Experiment and animal certificate number: SCXK (Shanghai) 2013-0016
Experimentation:
Before blood specimen collection, the 2M Fluorinse (esterase inhibition of 20L is added in EDTA-K2 anticoagulant tube in advance
Agent), after 80 degree of drying in oven, it is placed in 4 degree of refrigerator storages.
Rat, male, 187~197g of weight is randomly divided into 2 groups, is fasted in experiment noon before that day overnight but can
Free water, 4h is to food after administration.A group gives Odalasvir 3mg/kg, B group giving construction T-1 compound 3mg/kg, respectively
15min, 30min, 1,2,3,5,8,10h take blood 100-200L or so from rat orbital vein after administration, are placed in through EDTA-K2
It in the Eppendorf pipe of anticoagulant 0.5mL, mixes immediately, after anticoagulant, after being as early as possible gently mixed by inversion test tube 5-6 times, blood is taken
It is placed in ice chest after good, blood sample, centrifugal separation plasma, collection are complete under the conditions of 4000rpm, 10min, 4 DEG C in 30min
It is saved immediately in -20 DEG C after portion's blood plasma.The blood concentration in the blood plasma of each time point is measured after all time point sample acquisitions.
It is united using Winnonin software by non-chamber according to mean blood plasma concentration-time data after above-mentioned resulting administration
Meter square theory seeks calculation male SD rat, and i.g gives the medicine after Odalasvir (3mg/kg), formula T-1 compound (3mg/kg) respectively
For dynamics relevant parameter, see Table 3 for details.
Medicine is for parameter after 3 rat of table gives Odalasvir and formula T-1 compound
| PK parameter | Odalasvir | T-1 |
| Cmax(ng/mL) | 53865.5±1814.2 | 58141.1±8516.1 |
| t1/2(hr) | 7.64±1.31 | 7.98±0.31 |
| AUC0-t(hr*ng/mL) | 62819.7±3778.6 | 64653.8±3976.9 |
| AUC0-∞(hr*ng/mL) | 65019.9±3217.5 | 67373.6±4116.9 |
| MRT(hr) | 4.01±0.37 | 4.30±0.12 |
As shown in table 3, compared with Odalasvir, the inventors discovered that compound T-1 has more preferably than Odalasvir
Activity, and there is excellent pharmacokinetic property, therefore be more suitable for inhibiting the change of hepatitis C virus protein NS5A
Object is closed, and then is suitble to the drug of preparation treatment infection with hepatitis C virus.
It should be understood that these examples are only for illustrating the present invention and are not intended to limit the scope of the present invention, in embodiment not
The experimental method of actual conditions is indicated, usually according to normal condition, or according to the normal condition proposed by manufacturer.Unless in addition saying
Bright, otherwise parts and percentages are parts by weight and weight percent.
The above content is a further detailed description of the present invention in conjunction with specific preferred embodiments, and it cannot be said that
Specific implementation of the invention is only limited to these instructions.For those of ordinary skill in the art to which the present invention belongs, exist
Under the premise of not departing from present inventive concept, a number of simple deductions or replacements can also be made, all shall be regarded as belonging to of the invention
Protection scope.
Claims (5)
1. formula (I) compound represented or its pharmaceutically acceptable salt,
Wherein, R1、R2、R3、R4、R5And R6It is each independently hydrogen or deuterium；
R7、R8、R9、R10、R11、R12、R13、R14、R15、R16、R17、R18、R19、R20、R21、R22、R23、R24、R25、R26、R27、R28、
R29、R30、R31、R32、R33、R34、R35、R36、R37、R38、R39、R40、R41、R42、R43、R44、R45、R46、R47、R48、R49、R50、R51、
R52、R53、R54、R55、R56、R57、R58、R59、R60、R61、R62、R63、R64、R65、R66、R67、R68It is hydrogen；
Additional conditions are R1、R2、R3、R4、R5、R6In at least one be deuterium.
2. compound according to claim 1, it is characterised in that: the compound is following compounds or it pharmaceutically may be used
The salt of receiving:
3. a kind of pharmaceutical composition, it is characterised in that: it contains pharmaceutically acceptable carrier and as claim 1~2 is any
Compound described in one or its pharmaceutically acceptable salt.
4. pharmaceutical composition according to claim 3, it is characterised in that: it also includes other reactive compounds, the work
Property compound be immunomodulator or antiviral drugs.
5. a kind of compound as described in claim 1~2 any one is preparing for treating infection with hepatitis C virus
Purposes in drug.
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| CN201610017674.1A CN106083829B (en) | 2016-01-12 | 2016-01-12 | A kind of hepatitis C virus inhibitors, pharmaceutical composition and its application |
| PCT/CN2016/106875 WO2017121188A1 (en) | 2016-01-12 | 2016-11-23 | Hepatitis c virus inhibitor, pharmaceutical composition and application thereof |
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| CN106083829B (en) * | 2016-01-12 | 2019-01-22 | 深圳市塔吉瑞生物医药有限公司 | A kind of hepatitis C virus inhibitors, pharmaceutical composition and its application |
| CN110840969B (en) * | 2019-11-22 | 2021-04-16 | 南方医科大学 | Traditional Chinese medicine composition for treating hepatitis C and application thereof |
| CN115677595B (en) * | 2022-10-26 | 2024-06-14 | 江苏睿实生物科技有限公司 | Preparation method of 2,4, 5-trichloropyrimidine |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2008021927A2 (en) * | 2006-08-11 | 2008-02-21 | Bristol-Myers Squibb Company | Hepatitis c virus inhibitors |
| CN103561739A (en) * | 2011-05-27 | 2014-02-05 | 艾其林医药公司 | Subsituted aliphanes, cyclophanes, heteraphanes, heterophanes, hetero-heteraphanes and metallocenes useful for treating hcv infections |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2008021927A2 (en) * | 2006-08-11 | 2008-02-21 | Bristol-Myers Squibb Company | Hepatitis c virus inhibitors |
| CN103561739A (en) * | 2011-05-27 | 2014-02-05 | 艾其林医药公司 | Subsituted aliphanes, cyclophanes, heteraphanes, heterophanes, hetero-heteraphanes and metallocenes useful for treating hcv infections |
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| Title |
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| Deuterated drugs: where are we now？;Graham S Timmins;《Expert.Opin.Ther.Patents》;20141231;第24卷(第10期);第1-9页 |
| Deuterium Medicinal Chemistry: A New Approach to Drug Discovery and Development;Scott L. Harbeson等;《MEDCHEM NEWS》;20140530(第2期);第8-22页 |
| 氘代作用在药物研究中的应用;汪文峰等;《齐鲁药事》;20101231;第29卷(第11期);第682-684页 |
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| CN106083829A (en) | 2016-11-09 |
| WO2017121188A1 (en) | 2017-07-20 |
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